Isolation and identification of the bacterial isolates
A total of 27 unique colony types were isolated from the soil. Four strains (UR1, UR16, UR20 and UR21) were selected based on their urease activity. The 16S rRNA gene was sequenced and compared with the known sequence in the NCBI database. The results revealed that the isolates UR1, UR16, and UR21 are closely related to one another and correspond to the genus of Bacillus sp. and the isolate UR20 belongs to the genus Citrobacter sp. (Fig. S1). The obtained sequences of UR1, UR16, UR20 and UR21 were submitted to GenBank under the accession No. MT022031, MT022032, MT022034 and MT022035, respectively.
Biological characteristics of the bacterial strains
The toxicity of heavy metals is a serious aspect taken into account for the remediation process because the performance and existence of the bacteria in a contaminated environment predominantly depend on it (Nokman et al. 2019). Among the selected bacterial isolates, UR20 exhibited a high degree of resistance to Pb and the MIC value was 1400 mg L− 1, followed by the isolate UR21 showed the MIC value of 1300 mg L− 1. Whereas, the MIC value for the Cd was 500 mg L− 1 and 400 mg L− 1 for UR20 and UR21, respectively (Table 2). The increasing amount of heavy metal has a toxic effect on the growth of microorganisms. The ability of isolates to resist the different heavy metals provides an opportunity to use them in multiple metal-contaminated sites for bioremediation (Satyapal et al. 2018).
Table 2 Minimum inhibitory concentrations of heavy metals
|
MIC results
|
|
Isolates
|
Lead conc.
(mg L-1)
|
Cadmium conc.
(mg L-1)
|
UR 1
|
1100
|
500
|
UR 16
|
1200
|
400
|
UR 20
|
1400
|
500
|
UR 21
|
1300
|
400
|
MIC is defined as the minimum concentration of heavy metal that inhibits the bacterial growth.
The calcite production capability of urease-producing microorganism leads to the precipitation of soluble metal ions and finally convert them to carbonates (Gomaa et al. 2018). The highest calcite production was noticed in UR21 (20.2 mg mL− 1) followed by UR1 (20.2 mg mL− 1), UR20 (17.1 mg mL− 1) and UR16 (19.6 mg mL− 1) as shown in Fig. 1. The metabolic product (CO32−) was released by the microorganisms that react with (Ca2+) ion present in the medium and contribute to the precipitation of minerals (Dhami et al. 2013). The carbonate concentration was increased when calcium (Ca2+) and carbonate (CO32−) ions are prominent (Qian et al. 2010). Urease influences the chemical process associated with the formation of biominerals through four distinct parameters, including calcium concentration, cell surface, pH, and accessibility of nucleation sites (Anbu et al. 2016). The first two factors are responsible for the concentration of CO32− and the nucleation site helps in the production of calcium carbonate (Taylor et al. 2013). The cell surface of the bacteria is covered with negatively charged groups that function as a binding site for the divalent cations (e.g. Ca2+, Mg2+) and these nucleation sites lead to the deposition of calcite (Anbu et al. 2016). This research showed that the isolates were able to produce a high amount of calcium carbonates under suitable conditions due to their urease activity.
Urease plays a vital role in the formation of carbonate crystals in a media supplemented with calcium and urea (De Muynck et al. 2010). The urease activity of the four isolates was shown in (Fig. 1). The highest urease activity was observed by the isolate UR20 of 58.1 U mL− 1 followed by the isolates UR21, UR16 and UR1, with the urease activity of 55.2 U mL− 1, 39.9 U mL− 1 and 27.2 U mL− 1, respectively. It is commonly believed that the formation of calcium carbonate depends on enzyme activity, a high rate of urease activity accelerates the production of calcium carbonate (Al-salloum et al. 2016). The ability of bacterial strains to hydrolyze urea is affected by many factors including temperature, pH, incubation period and concentration of urea, and by providing a suitable condition to a specific strain the enzymatic activity of that strain can be enhanced (Sheng et al. 2020).
Urease activity of the bacterial isolates under different environmental conditions
The isolates grown under different temperatures have shown different urease activity. The maximum ability to hydrolyzed urea was observed when the bacterial isolates was incubated at temperatures 28°C and 32°C (Fig. 2(a)). The ability of the bacterial strains to hydrolyzed urea was comparatively low when incubated at the temperature of 24°C. These results indicated that the enzymatic activity of the bacterial strains could be influenced by the temperature. The appropriate temperature to get maximum enzyme activity varies from 24 to 37°C (Soon et al. 2014). Imran et al. (2019) demonstrated that the enzyme activity of the isolate rises 5.0 to 10 times when the temperature raised from 10 to 20°C. Consequently, based on these experimental results, our study indicated that temperature was one of the crucial factors that influence the urease activity because the amount of urea hydrolyzed varies with temperature.
The results obtained after growing isolates under different pH revealed that the bacterial strains exhibit the highest ability to hydrolyzed urea at the pH of 7.5 and 8.0. The urease activity of the bacterial isolate is relatively low at the pH of 6.5 as shown in Fig. 2(b). The heterotrophic facultative and aerobic bacteria which are widely used in MICP known to grow well under weak alkaline conditions (Sheng et al. 2020). The investigation shows that the strain Bacillus megaterium and B.cereus can grow well at the pH range from 6.5 to 11.5. whereas, some strains like B. subtilis and B. thuringiensis can grow at the pH range from 6.0 to 10 (Kaur et al. 2013). The pH has not only affects the urease activity but also affects the metabolism of bacteria. Our investigations showed that the initial pH of the medium plays an important role to attain maximum ureases activity.
Removal of Pb and Cd in solution
The addition of bacterial isolates in YA broth containing Pb and Cd supplemented with urea showed high removal efficiency of heavy metals after 72 h of incubation. The highest removal rate for Pb reached to 81.9% and 77.1% by the isolate UR21 and UR20, respectively (Fig. 3(a)). Similarly, in the case of Cd, UR21 had the highest removal rate of 65.0% (Fig. 3(b)). It was observed that the removal rate of Pb and Cd become stable from 60 to 72 h. It is likely that the metal ion occupied all the available binding sites and the functional group provided by the bacteria, thus, there are no more available sites for the metal ion to bind (Zhao et al. 2019). Our studies and the previous research revealed that the deposition rate of Pb is higher as compared to Cd (Rahman et al. 2019). Heavy metals have different sizes of ionic radius and the variation in the ionic radius of these elements could be responsible for the diversity in the precipitation. The ionic radius of Pb is higher than Cd so it might be one of the reasons that the removal rate of Pb in the solution is higher (He et al. 2019). The remediation rate of heavy metals mainly depends on the number of bacterial cells. The increase in growth of the bacteria promoted the production of urease enzyme, which enhanced the removal rates of the heavy metals (Kang et al. 2015; Li et al. 2008). The Pb and Cd ions present in the solution attached with the available binding sites (such as carboxyl, amino, and phosphate) of the bacterial cell and then utilized the carbonates produced by the hydrolysis of urea to generate lead and cadmium carbonates, which convert these soluble metals into insoluble form and reduced their toxic level by lowering bioavailability (Zhao et al. 2017). Zhao et al. (2019) reported that free Cd ion present in soil incorporates with the carbonate ion produced by the ureolytic isolate to form CdCO3 precipitates. The present study demonstrated that the strain UR21 has great potential in immobilization of Pb and Cd.
SEM-EDS and XRD analysis of the precipitates
The precipitates obtained after the remediation process was observed under SEM to understand the different morphological characteristics. The surface of Pb and Cd carbonate crystals had several bacterial imprints, which demonstrated that the bacteria surface act as a nucleation site for the precipitation of carbonates. As illustrated in the Fig. 4, the PbCO3 crystals produced during bacterial ureolysis were needle-shaped which are also consistent with the findings of Kang et al. (2015). Whereas, the CdCO3 crystals produced were roughly rhombohedral in shape (Fig. 4). The variation in the morphology of the crystals formed is based on the urease activity of the bacteria and the bacterial species (Park et al. 2010). The EDS analysis of the precipitates indicated the existence of lead (Pb), carbon (C) and oxygen (O) in lead carbonate crystals. Similarly, cadmium (Cd), carbon (C) and oxygen (O) were detected in cadmium carbonate crystals. The analysis by SEM and EDS evidently revealed that the isolates could efficiently transform the soluble Pb and Cd into insoluble PbCO3 and CdCO3 crystals and thus promoted the remediation of Pb and Cd.
XRD analysis also indicated that the carbonate precipitation was accelerated by the MICP process (Fig. 4). The XRD spectra showed an increment of calcium carbonate peaks in the form of vaterite and calcite. More importantly, XRD spectra verified that the strain UR 21 immobilized Pb and Cd specifically in the form of lead carbonate and cadmium carbonate, which has low toxicity as compared to Cd ions. In contrast to PbCO3, the peaks of CdCO3 were slightly low. Similar results were also reported by other scholars (Chen and Achal 2019; Kang et al. 2016). These findings revealed that Pb2+ and Cd2+ with an ionic radius similar to Ca2+ was introduced into the CaCO3 crystal by replacing Ca2+ in the lattice or accessing the interstices (Achal et al. 2011). Furthermore, Pb2+ and Cd2+ ions may be adsorbed to the surface of calcite through its lattice (Zhu et al. 2016).
Fractionation of Pb and Cd in soil
The mobility and toxicity of the Pb and Cd in the environment was estimated by their fractions. The results revealed that different treatments had a significant effect on the Pb and Cd fractions in soil. The prevalent fraction of Pb in the control was exchangeable-Pb (64.64%), followed by the carbonated-Pb fraction (16.7%), while the Fe-Mn oxide-Pb, organic matter-Pb and the residual-Pb was accounted for 10.6%, 4.12% and 3.91%, respectively. The exchangeable-Pb fraction in soil decreased to varying degrees after 40 days of incubation, revealing that the exchangeable fraction of Pb was transform into other fractions as illustrated in Fig. 5(a). As compared to control, the proportion of soluble-exchangeable Pb was declined to 19.5%-43.8% in all treated groups and the proportion of carbonated-Pb was enhanced to 18.5%-35.9%. Meanwhile, the proportion of Fe-Mn oxide-Pb was increased to 1.56%-6.69% in all treated groups except S4 group whereas, the proportion of organic-Pb and residual-Pb in soil was less than 6%. A similar pattern was found in the case of Cd, the dominant fraction in the control group was exchangeable-Cd (70.68%), followed by carbonated-Cd (11.01%), while the Fe-Mn oxide-Cd, organic matter-Cd and the residual-Cd was comprises for 8.37%, 4.17% and 5.78%, respectively (Fig. 5(b)). The exchangeable-Cd was reduced to 20.22%-37.78% and the carbonated-Cd was increased to 17.61%-36.71% in other treatments. Whereas, as compared to control group, no significant difference in Fe-Mn oxide-Cd, organic matter-Cd and the residual-Cd were observed, which are in accordance with the findings of Ren et al. (2020).
It's worth mentioning that both the exchangeable Pb and Cd content dramatically decreased with the addition of UR 21, especially with the treatment of UR21 + urea + EGS in the S4 group, revealing the pivotal role of UR21 in the MICP pathway. Based on the fractional variations in Pb and Cd exchangeable and carbonated forms, it could be concluded that during the MICP process, converting the soluble-exchangeable to carbonated bound Pb/Cd was the key strategy for reducing HMs toxicity and mobility. This trend of fraction variation was also observed in earlier reports (Chen and Achal 2019; Zhu et al. 2016). The minimal association between UR21 and Fe-Mn oxide proportion of Pb and Cd could be due to the reduced bioavailability (Krishnamoorthy et al. 2006). Moreover, the possible reason behind the unchanged fraction of residual-bound Pb and Cd is that it is tightly bound and very stable under natural conditions (Zhao et al. 2019).
DTPA available Pb and Cd
The content of DTPA available Pb and Cd in the treated soil was declined as the incubation time increased. According to Figure (6a,b), compared with the control, the S4 treatment reduced the DTPA-extractable Pb and Cd contents in the soil by 29.2% and 25.2% after 40 days of incubation. At the same time, the S3 group also exhibited a prominent reduction in DTPA-extractable Pb (by 27.1%) and Cd (by 20.6%). The results suggested that the immobilization ability of available Pb is slightly higher than the available Cd. Meanwhile, it can be seen in Fig. 6a,b that the immobilizing efficiency of strain UR21 combine with EGS was relatively high as compared to strain UR21 combined with Urea. The following reason may be contributed to achieving those outcomes. Firstly, the EGS contain many substances involving amino acid that had functional group similar to urea, which could be used by urease (Nagamalli et al. 2017). Secondly, the enrichment of calcium carbonate source provided for the ureolytic bacteria by the addition of EGS to the soil, triggered the MICP process (Peng et al. 2020). Therefore the outcome revealed that the EGS can be combined with urease-producing bacteria as an alternate of urea, to achieve high immobilization efficiency in the soil.
Effect of MICP on soil pH and CEC
The pH and CEC values of the soil were assessed to analyze the effect of MICP on the physicochemical properties of soil. The soil pH gradually increased during the period of 40 days incubation (Fig. 7(a)), The soil pH in S4 group increased to 0.41 units followed by S3, S2 and S1 group (0.36, 0.07 and 0.06 units) respectively. However, as compared to control group, the CEC value of the soil was 35.41 cmol kg− 1 in S3 group followed by S4 group which was 33.91 cmol kg− 1 after 40 days of incubation (Fig. 7(b)). Our results indicated that as compared to the UR21 alone treatment, the combination of UR21 with urea and EGS significantly enhanced the soil pH. The rise in pH is attributed largely to the presence of eggshells, most of which is composed of CaCO3. The possible reason could be the neutralization reaction between carbonate and H+ in an acidic environment in the presence of eggshells (Luo et al. 2018). In addition, the elevation of pH could be due to the generation of NH4+, formed by the decomposition of urea during the MICP process (Zhu et al. 2016). CEC is an indicator of nutrient abundance in the soil. The result suggested that the application of EGS improves the soil CEC value, which is consistent with the findings of Yong et al. (2010). The increase in the CEC in the presence of EGS could be due to the enrichment of Ca2+ which is consistent with the findings of Peng et al. (2020). The elevation of CEC in soil plays a vital role in the stabilization of heavy metals, the ions of the heavy metal are exchanged with Mg2+, Na+, and other cations thus get precipitated in the soil (Bolanle-ojo et al. 2014).
Effect of MICP on soil enzymatic activity
Heavy metal pollution may have a detrimental effect on the biological activities of soil microbes (Pan and Yu 2011). The urease and catalase activity of soil are sensitive biomarker of heavy metals. Hence, they are employed to determine the impact of soil amendments on biological functions of soil (He et al. 2019; Shi and Ma 2017). The effects of MICP on enzymatic activity in the contaminated soil are diverse, and possibly correlated with the biological activities of the bacteria and soil environmental conditions (Peng et al. 2020). The change in soil urease and catalase activity after 40 days of incubation was illustrated in Fig. 8. The results indicated that the soil urease activity was promoted most in S2 group, increased by 120% followed by S4 treatment with an increment of 82%, respectively as compared to control. The possible reason could be the addition of urea may contribute to enhance the urease activity in the S2 and S4 group. Besides, the S4 treatment has the maximum carbonated bound Pb and Cd along with the urease activity, but the least exchangeable Pb and Cd fraction (Fig. 5). Which also indicated that Cd passivation was strongly influenced by soil UR21, which generate urease to break down urea. As a result of this, Cd precipitates as carbonate. Earlier report also showed that the urease, a main enzyme involved in the MICP pathway (Chen and Achal 2019), not only regulates the heavy metal immobilization in soil, but it is also closely linked to the geochemical mechanism of the N-cycle (Wang et al. 2017).
In contrast to urease activity, the results suggested that the soil catalase activity was significantly high in S4 group, increased by 71% followed by S2 treatment with an increment of 50%, respectively as compared to control. Although catalase does not play an important role in the MICP process, but it serves as an essential indicator to evaluate the activation of soil microbes (Lemanowicz 2019).